Tumor Uptake of Triazine Dendrimers Decorated with Four, Sixteen, and Sixty-Four PSMA-Targeted Ligands: Passive versus Active Tumor Targeting

Abstract

Various glutamate urea ligands have displayed high affinities to prostate specific membrane antigen (PSMA), which is highly overexpressed in prostate and other cancer sites. The multivalent versions of small PSMA-targeted molecules are known to be even more efficiently bound to the receptor. Here, we employ a well-known urea-based ligand, 2-[3-(1,3-dicarboxypropyl)-ureido] pentanedioic acid (DUPA) and triazine dendrimers in order to study the effect of molecular size on multivalent targeting in prostate cancer. The synthetic route starts with the preparation of a dichlorotriazine bearing DUPA in 67% overall yield over five steps. This dichlorotriazine reacts with G1, G3, and G5 triazine dendrimers bearing a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group for ⁶⁴Cu-labeling at the core to afford poly(monochlorotriazine) intermediates. Addition of 4-aminomethylpiperidine (4-AMP) and the following deprotection produce the target compounds, G1-(DUPA)₄, G3-(DUPA)₁₆, and G5-(DUPA)₆₄. These targets include 4/16/64 DUPA groups on the surface and a DOTA group at the core, respectively. In vitro cell assay using PC3-PIP (PSMA positive) and PC3-FLU (PSMA negative) cells reveals that G1-(DUPA)₄ has the highest PC3-PIP to PC3-FLU uptake ratio (10-fold) through the PSMA-mediated specific uptake. While G5-(DUPA)₆₄ displayed approximately 12 times higher binding affinity (IC₅₀ 23.6 nM) to PC3-PIP cells than G1-(DUPA)₄ (IC₅₀ 282.3 nM) as evaluated in a competitive binding assay, the G5 dendrimer also showed high non-specific binding to PC3-FLU cells. In vivo uptake of the ⁶⁴Cu-labeled dendrimers was also evaluated in severe combined inmmunodeficient (SCID) mice bearing PC3-PIP and PC3-FLU xenografts on each shoulder, respectively. Interestingly, quantitative imaging analysis of positron emission tomograph (PET) displayed the lowest tumor uptake in PC3-PIP cells for the midsize dendrimer G3-(DUPA)₁₆ (19.4 kDa) (0.66 ± 0.15%ID/g at 1 h. p.i., 0.64 ± 0.11%ID/g at 4 h. p.i., and 0.67 ± 0.08%ID/g at 24 h. p.i.). Through the specific binding of G1-(DUPA)₄ to PSMA, the smallest dendrimer (5.1 kDa) demonstrated the highest PC3-PIP to muscle and PC3-PIP to PC3-FLU uptake ratios (17.7 ± 5.5 and 6.7 ± 3.0 at 4 h p.i., respectively). In addition, the enhanced permeability and retention (EPR) effect appeared to be an overwhelming factor for tumor uptake of the largest dendrimer G5-(DUPA)₆₄ as the uptake was at a similar level irrelevant to the PSMA expression.

Keywords: 64Cu; CT; DUPA; EPR; PC3-FLU; PC3-PIP; PET Imaging; dendrimer; prostate cancer; prostate-specific membrane antigen (PSMA); triazine; tumor.

Chart 1

Schematic Design of Triazine Dendrimers bearing DUPA ligands on the Surface and a DOTA at the Core.

Scheme 1

Synthesis of DUPA-DCT.

Scheme 2

Synthesis of G1-(DUPA)₄, G3-(DUPA)₁₆, and G5-(DUPA)₆₄.

Figure 1

¹H NMR spectra of DUPA-DCT, G5 Platform, and 6 in the distinctive region for monitoring reaction of the DCT with the deprotected G5 dendrimer. The theoretical integration value of 6, which appeared at 1.75 ppm, is supposed to be 556.

Figure 2

In vitro cell assays. (A) Internalization of the ⁶⁴Cu-labeld dendrimers in PC3-PIP cells. (B) The cellular uptake ratio of the ⁶⁴Cu-labeld dendrimers in PC3-PIP (PSMA⁺) and PC3-FLU (PSMA⁻) cells (n = 3).

Figure 3

The PSMA binding affinities of G1-(DUPA)₄, G3-(DUPA)₁₆, and G5-(DUPA)₆₄ were evaluated using PC3-PIP cells and a ¹²⁵I-labeled PSMA radioligand as the PSMA-specific competitor. The IC₅₀ values of G1-(DUPA)₄, G3-(DUPA)₁₆, and G5-(DUPA)₆₄ were calculated to be 23.6 nM (R² = 0.95), 226.7 nM (R² = 0.94), and 282.3 nM (R² = 0.88), respectively (n = 4).

Figure 4

PC3-PIP to muscle and PC3-PIP to PC3-FLU uptake ratios of the ⁶⁴Cu-labeled dendrimers in SCID mice bearing PC3-PIP cells on the left shoulder and PC3-FLU cells on the right shoulder at 1 h p.i. (A), 4 h p.i. (B), and 24 h p.i. (C). (n = 3 for G1-(DUPA)₄ and G5-(DUPA)₆₄; n = 4 for G3-(DUPA)₁₆).

Figure 5

Representative trans-axial PET-CT images of the ⁶⁴Cu-labeled dendrimers in SCID mice bearing PSMA⁺ PC3-PIP xenografts on the left shoulder and PSMA^-PC3-FLU xenografts on the right shoulder at 1, 4, and 24 h p.i. (n = 3 for G1-(DUPA)₄ and G5-(DUPA)₆₄; n = 4 for G3-(DUPA)₁₆).